Platelet alloantigens are substances that induce the production of alloantibodies when platelets bearing such antigens are infused into patients who lack the specific alloantigen. Immune responses to platelet alloantigens are involved in the pathogenesis of several clinical syndromes including neonatal alloimmune thrombocytopenia, post-transfusion purpura, and refractory responses to platelet transfusion. In addition, immune thrombocytopenia can be an unusual complication of a type of graft-versus-host disease in which donor lymphocytes make alloantibodies specific for the platelets produced by the recipient of an organ allograft.
Patients can lack a particular platelet-associated antigen altogether because they have defective alleles of the gene encoding the antigen. Such patients can make antibodies against platelets of virtually all donors that bear the platelet-associated antigen. For example, patients with Bernard-Soulier syndrome, who lack platelet GPIb-V-IX, or patients with Glanzmann thrombasthenia, who lack expression of GPIIb (CD41) and GPIIIa (CD61), can be induced to make broadly-reactive antiplatelet antibodies. Also, several percent of Japanese and approximately 0.3 percent of Caucasians are deficient in CD36, one of the major platelet glycoproteins of platelets that also is known as GPIV. Because these patients lack a platelet antigen, they can develop antiplatelet antibodies specific for the deficient platelet protein after receiving transfusions of platelets from normal donors or after pregnancy. More commonly, platelet-specific alloantigens result from genetic polymorphism in genes encoding functional platelet proteins. These alloantigens first were defined by antiplatelet antibodies discovered in the sera of multiparous females who gave birth to infants with neonatal thrombocytopenia. Many of these subsequently were found to recognize allotypic determinants of platelet-associated membrane glycoproteins, such as GPIIb/IIIa (CD41/CD61). Each of these allotypic determinants may be generated by only a single amino acid substitution in a major platelet-associated glycoprotein. However, it is possible that glycosylation may contribute to or influence the expression of certain Human Platelet Alloantigenic (HPA) epitopes, such as those associated with human platelet antigen 3 (HPA-3). In any case, these amino acid substitutions generally do not appear to affect the function of platelets in vitro. However, it is conceivable that the genetic polymorphism in platelet glycoproteins may be associated with more subtle differences in platelet physiology that can contribute to the relative risk for thrombosis and/or atherosclerosis. (Williams Hematology, Chapter 138)
The human leukocyte histocompatibility antigens, HLA, are polymorphic cell surface glycoproteins that present antigen peptide fragments to T-cell receptors. HLA antigens are encoded by multiple, closely linked genes, located in a 4-Mb region of DNA on chromosome 6, that comprise the major histocompatibility complex (MHC) and play a central role in the regulation of immune responses. In general, the MHC genes are inherited as a single unit in simple Mendelian fashion. The products of the MHC HLA-A, HLA-B, and HLA-C genes are called class I antigens. Class I antigens are expressed on essentially all tissues in the body and present small peptide fragments to CD8+ T cells. (Williams Hematology, Chapter 138)
There are six major groups of HLA antigens: HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DQ, and HLA-DP. These groups are divided into classes of antigens designated as class I and class II, representing the two types of HLA molecules. The HLA-A, HLA-B, and HLA-C antigens are the class I antigens. The HLA-DR, HLA-DQ, and HLA-DP antigens are the class II antigens. (Williams Hematology, Chapter 138)
In addition to the HLA antigens, platelets also express glycoproteins that can be recognized by autoantibodies or by antibodies made by recipients of platelet transfusions. The latter are due to platelet alloantigens that reflect polymorphism in the genes encoding major platelet glycoproteins. Immune responses to platelet alloantigens are involved in the pathogenesis of several clinical syndromes, including neonatal alloimmune thrombocytopenia, post-transfusion purpura, and refractory responses to platelet transfusion. (Williams Hematology, Chapter 138)
The inventors have discovered a method of creating human platelets expressing specific HPA isotypes utilizing CRISPR/Cas9 gene editing methods and laboratory cell culture techniques. Deletion of the β2 microglobulin gene offers distinct practical advantages that will be outlined in the description of the invention.
The inventors have discovered a method to generate human platelets that express any minor or major HPA that is desired, so called “designer platelets”. After demonstrating that one can convert PlA1 to PlA2 in DAMI cells, the inventors have most recently shown this conversion in human induced pluripotent stem (iPS) cells which can be differentiated into megakaryocytes and then platelets using methods known in the art. Our initial anticipated use will be the development of a new platform for rapid flow cytometric detection of rare platelet antigens. This will be made useful and easier than antigen capture ELISA test (ACE) or modified antigen capture ELISA test (MACE) because we will also knock out β2 microglobulin in the iPS cells so that anti-HLA antibodies in maternal or patient sera will have no Class I targets to bind to, hopefully simplifying the assay and lowering back-ground.
In concept, it would be very useful to have such a panel for laboratory testing. Even though the market might be small, you could argue that the project might provide proof-of principal for future studies to express rare RBC antigens (of which there are many). Right now, reference blood banks maintain frozen RBC panels expressing various low frequency RBC antigens or (equally important) lacking high frequency (public) antigens and they thaw them out when they need to check specificity of an unknown antibody in a patient. Producing “designer RBCs”, that look identical to physiologic RBC's, could be a serious technical challenge because the cultured cells need to shed their nucleus, among other things and techniques to do this are not currently completely finalized. However, one could express the rare RBC antigens in nucleated RBC's, anucleated RBC's, platelets, iPS cells, or iPS cell and then use these cell types as laboratory controls and sources of these rare antigens.
An additional use of iPS—derived designer platelets will be to provide rare platelet types for transfusion. This will require the use of a platelet bioreactor. The commercial use of platelet bioreactors is not yet commonplace. However, one advantage of this strategy, is the gene editing arm of the technology, which allows you to make platelets of specific HPA types. The therapeutic use of platelets that lack specific HLA antigens or express matching HLA antigens could be a solution to various forms of platelet refractoriness. The platelets would be group ABO negative or group O, to rule out issues with ABO compatibility. HPA-1a-negative platelets might be useful for the most common form of NAIT. Platelets matched for other HPA antigens are occasionally useful in immunized thrombocytopenic patients.